I have seen procedures in a reputable lab where they do indeed pick multiple colonies and mix them; the people with the most experience claim(ed) that this MUST be done for the particular prep they were doing or else they get very low yields. The concern would be (in my mind) that if the different clones do not express exactly the same protein, but for example truncations, then you are in big trouble. But apparently that is not the case and the differences are in some other part of the little critter that expresses protein. I'd much rather make different cultures, each based on a single colony and see which works best. It makes somewhat sense that they might not all be exactly equal, but the standard protocol to get one, presumably homogeneous colony as a start makes good sense too.
Mark -----Original Message----- From: Andreas Förster <[EMAIL PROTECTED]> To: CCP4BB@JISCMAIL.AC.UK Sent: Sat, 2 Feb 2008 9:51 am Subject: Re: [ccp4bb] Codon Optimized Expression There has been a somewhat related discussion in the lab the other day. If some colonies might express well and other not so well, why don't I just scoop up loads and start my culture from them? This way I'll be more certain to get the clones that express. The clones that don't express will dilute the yield, but I doubt they'd outcompete the rest, would they? For me, the start-with-plenty procedure has always worked well, but some insist on starting from single colonies. Andreas James Stroud wrote: > On Feb 2, 2008, at 2:15 AM, M T wrote: >> One classical way to optimize expression level is to screen culture >> conditions. >> >> For my proteins, I solved my expression problems by changing the >> expression vector to a pET or changing a pET 20 to a pET 30 (if the >> protein is toxic). >> >> But keep in mind that a low but folded expression is better than a >> high expression to inclusion body. > > Anecdotal evidence also suggests to try picking several colonies from > the original cloning procedure and doing test expressions with each. > Some clones express better than others even though they should in > principle express identically. I have no idea what the mechanism for > this differential expression could be. > > -- > James Stroud > UCLA-DOE Institute for Genomics and Proteomics > Box 951570 > Los Angeles, CA 90095 > > http://www.jamesstroud.com > ________________________________________________________________________ More new features than ever. Check out the new AIM(R) Mail ! - http://webmail.aim.com
